Genome Analysis of Pseudomonas aeruginosa Strains from Chronically Infected Patients with High Levels of Persister Formation

Abstract

The appearance of persister cells with low metabolic rates are key factors leading to antibiotic treatment failure. Such persisters are multidrug tolerant and play a key role in the recalcitrance of biofilm-based chronic infections. Here, we present the genomic analyses of three distinct Pseudomonas aeruginosa Egyptian persister-isolates recovered from chronic human infections. To calculate the persister frequencies, viable counts were determined before and after treatment with levofloxacin. The susceptibilities of isolates to different antibiotics were determined using the agar-dilution method. To determine their recalcitrance, the levofloxacin persisters were further challenged with lethal concentrations of meropenem, tobramycin, or colistin. Furthermore, the biofilm formation of the persister strains was estimated phenotypically, and they were reported to be strong biofilm-forming strains. The genotypic characterization of the persisters was performed using whole genome sequencing (WGS) followed by phylogenetic analysis and resistome profiling. Interestingly, out of the thirty-eight clinical isolates, three isolates (8%) demonstrated a persister phenotype. The three levofloxacin-persister isolates were tested for their susceptibility to selected antibiotics; all of the tested isolates were multidrug resistant (MDR). Additionally, the P. aeruginosa persisters were capable of surviving over 24 h and were not eradicated after exposure to 100X-MIC of levofloxacin. WGS for the three persisters revealed a smaller genome size compared to PAO1-genome. Resistome profiling indicated the presence of a broad collection of antibiotic-resistance genes, including genes encoding for antibiotic-modifying enzymes and efflux pump. Phylogenetic analysis indicated that the persister isolates belong to a distinct clade rather than the deposited P. aeruginosa strains in the GenBank. Conclusively, the persister isolates in our study are MDR and form a highly strong biofilm. WGS revealed a smaller genome that belongs to a distinct clade.

Miconazole and phenothiazine hinder the quorum sensing regulated virulence in Pseudomonas aeruginosa

“…This pathogen has an extraordinary capacity to infect different body parts, including invasive infections, which lead to lengthy hospital stays and high mortality [ 37 ]. Further, P. aeruginosa has developed multiple drug resistance (MDR) and persister strains, which strongly limits the treatment options and represent a major threat worldwide [ 38 , 39 ]. A recent strategy, used to overcome bacterial resistance, targets virulence factors instead of killing the bacteria.…”

Multifunctional Spirogyra-hyalina-Mediated Barium Oxide Nanoparticles (BaONPs): Synthesis and Applications

“…Turbidity of the overnight cultures was adjusted to the No. 0.5 McFarland Standard [58,59], according to CLSI (Clinical Laboratory and Standard Institute) guidelines.…”

Antibacterial and Antibiofilm Activity of Ficus carica-Mediated Calcium Oxide (CaONPs) Phyto-Nanoparticles

“…The plates were allowed to remain in an incubator at 37 • C for 24 h. After incubation, zones of inhibition were measured in mm, according to [57][58][59] The CaONPs' antibiofilm potential was tested against E. coli, P. aeruginosa, S. aureus, K. pneumoniae, and P. vulgaris. The activity was performed in a 96-well microtiter plate, according to [60,61]. The wells were supplied with 100 µL of 0.5 OD bacterial suspension and 60 µL of CaONPs followed by incubation at 37 • C for 24-72 h. After incubation, the crystal-violet assay was used to quantify the biofilm.…”